Maize cytoplasmic male sterility (CMS) S-type restorer Rf3 gene, molecular markers and their use

ABSTRACT

The present disclosure provides a method for selecting a plant comprising a functional restorer gene for maize S-type cytoplasmic male sterility comprising the steps of (a) screening a population of plants for at least one marker nucleic acid, wherein the marker nucleic acid comprises an allele linked to the functional restorer gene for maize S-type cytoplasmic male sterility; (b) detecting the marker nucleic acid; (c) identifying a plant comprising the marker nucleic acid; and (d) selecting the plant comprising the marker nucleic acid, wherein the plant comprising the marker nucleic acid further comprises the functional restorer gene for maize S-type cytoplasmic male sterility. The present disclosure also provides methods for restoring fertility in a progeny of an S-type cytoplasmic male sterile plant and methods for transferring an Rf3 gene into a progeny plant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisional Application Ser. No. 61/922,349, filed on Dec. 31, 2013, theentire disclosure of which is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“73721_ST25.txt”, created on Dec. 30, 2014, and having a size of 25kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

The subject disclosure relates to plant fertility genes. In someembodiments, the disclosure relates to Rf3, a maize restorer offertility gene. In particular embodiments, the disclosure relates tocompositions and methods for restoring fertility to S-type cytoplasmicmale sterility (CMS), for example, by using molecular markers linked to,or residing within, the Rf3 gene. Particular embodiments relate tomethods for using particular nucleic acid sequences to identify plantsthat contain restorer of fertility to CMS-S, and methods for hybrid seedproduction.

BACKGROUND

The development of hybrid plant breeding has allowed for considerableadvances in quality and quantity of crops that are produced. Increasedyield and the combination of desirable characteristics, such asresistance to disease and insects, heat and drought tolerance, andvariations in plant composition are all possible, in part, due tohybridization procedures. Hybridization procedures rely on thecontribution of pollen from a male parent plant to a female parent plantin order to produce resulting hybrids.

Plants may self-pollinate if pollen from one flower is transferred tothe same or a different flower of the same plant. Alternatively, plantsmay cross-pollinate if the pollen originates in a flower from adifferent plant. Maize plants (Zea mays) may be bred using bothself-pollination and cross-pollination techniques. Maize plants havemale flowers, which are located on the tassel, and female flowers, whichare located on the ear of the same plant. Natural pollination in maizeoccurs when pollen from the tassels reaches the silks that are found atthe tops of the incipient ears. Importantly, the development of maizehybrids relies upon male sterility systems.

The development of maize hybrids requires the development of homozygousinbred lines, the crossing of these lines, and the evaluation of theresultant crosses. Pedigree breeding and recurrent selection are twobreeding methods that may be used to develop inbred lines from maizepopulations. Breeding programs combine desirable traits from two or moreinbred lines or various broad-based sources into breeding pools fromwhich new inbred lines are developed by selfing and selection of desiredphenotypes. A hybrid maize variety is the cross of two such inbredlines, each of which may have one or more desirable characteristicsabsent in one, or complementing the other. The new inbred plants arecrossed with other inbred lines and the resultant hybrids from thesecrosses are evaluated to determine which are more desirable. The hybridprogeny from the first generation are designated F₁. In the developmentof hybrids, only the F₁ hybrids are sought. The F₁ hybrid is typicallymore vigorous than its inbred parents. This hybrid vigor, termed“heterosis,” typically leads to more desirable traits, for example,increased vegetative growth and increased yield.

Hybrid maize seed can be produced by a male sterility systemincorporating manual detasseling. To produce hybrid seed, the tassel isremoved from the growing female inbred parent, which can be proximatelyplanted in various alternating row patterns with the male inbred parent.Consequently, provided that there is sufficient isolation from foreignmaize pollen, the ears of the female inbred will be fertilized only withpollen from the male inbred. The resulting seed is termed hybrid F₁seed.

However, manual detasseling is labor-intensive and costly. Manualdetasseling is also often ineffective because in some instancesenvironmental variation in plant development can result in plantstasseling after manual detasseling of the female parent plant iscompleted or because a detasseler might not completely remove the tasselof a female inbred plant. If detasseling is ineffective, the femaleplant will successfully shed pollen and some female plants will beself-pollinated. This will result in seed of the female inbred beingundesirably harvested along with the hybrid seed which is normallyproduced. Female inbred seed is not as productive as F₁ seed. Inaddition, the presence of female inbred seed can represent a germplasmsecurity risk for the producer of the hybrid seed.

A female inbred plant can also be mechanically detasseled by a machine.Mechanical detasseling is approximately as reliable as hand detasseling,but is faster and less expensive. However, most detasseling machinesproduces more damage to the plants than hand detasseling. Thus, neithermanual nor mechanical detasseling is entirely satisfactory at thepresent time.

Genetic male sterility is an alternative method that may beadvantageously used in hybrid seed production. The laborious detasselingprocess can desirably be avoided in some genotypes by using cytoplasmicmale-sterile inbred plants. In the absence of a fertility restorer gene,plants of a cytoplasmic male-sterile inbred are male sterile as a resultof factors resulting from the cytoplasmic, as opposed to the nuclear,genome. Therefore, the characteristic of male sterility is inheritedexclusively through the female parent in maize plants, since only thefemale provides cytoplasm to the fertilized seed. Cytoplasmicmale-sterile plants are fertilized with pollen from another inbred plantthat is not male-sterile. Pollen from the second inbred plant may or maynot contribute genes that make the hybrid plants male-fertile.Typically, seed from detasseled normal maize and cytoplasmicmale-sterile-produced seed of the same hybrid must be blended to ensurethat adequate pollen loads are available for fertilization when thehybrid plants are grown and to ensure cytoplasmic diversity.

Drawbacks to use of cytoplasmic male sterility (CMS) as a system for theproduction of hybrid seed include the association of specific variantsto CMS with susceptibility to certain crop diseases. See, e.g., Beckett(1971) Crop Science 11:724-6. This problem has specifically discouragedthe use of the CMS-T variant in the production of hybrid maize seed, andhas had a negative impact on the use of CMS in maize in general.

Cytoplasmic male sterility is the maternally inherited inability toproduce functional pollen. More than 40 sources of CMS have been foundand classified into three major groups by differential fertilityrestoration reactions. These groups are designated as CMS-T (Texas),CMS-S (USDA), and CMS-S (Charrua) (Beckett, 1971). In the CMS-T group,two dominant genes, Rf1 and Rf2, which are located on chromosomes 3 and9, respectively, are required for the restoration of pollen fertility(Duvick, 1965). The S-cytoplasm is restored by a single gene, Rf3, whichhas been mapped on chromosome 2 (Laughnan and Gabay, 1978).

In maize, the restorer of the S type of CMS behaves as a gametophytictrait. Maize plants with S cytoplasm are restored by the single dominantgene, Rf3, which was mapped to the long arm of chromosome 2 and locatedbetween the whp1 and bnl17.14 loci (Kamps and Chase, 1997). Tie (2006)reported that Rf3 was associated with SSR markers umc1525 and bn1g1520at distances of 2.3 and 8.9 cM, respectively. Zhang et al. (2006)identified three amplified fragment length polymorphism markers thatwere tightly linked to the Rf3 gene.

Heterozygous (Rf3/rf3) CMS-S plants are semi-fertile, sheddingapproximately 50% abortive collapsed pollen containing the rf3 alleleand 50% starch-filled fertile pollen containing the Rf3 allele. The rf3allele in Rf3/rf3 plants cannot be transferred to progeny throughsterile pollen, thus generating sterile plants in F2 generation (Tie etal., 2006). This type of inheritance makes it very difficult to collectaccurate phenotypic data from an F2 mapping population. Traditionalmethods for identifying mutations are labor and time-intensive,whole-genome sequencing was considered as an approach to determine thedifferences between CMS-S and restorer lines. At the same time, abackcross 1 (BC1) mapping population was designed to evaluate theidentified mutations. A BC1 mapping population is advantageously moreuseful to evaluate phenotypes. Individuals from a backcross populationhave either Rf3/rf3 or rf3/rf3 genotypes, and thus there is no need todistinguish fully fertile phenotype from partially fertile phenotypeduring the phenotyping process.

Molecular markers are particularly useful for accelerating the processof introducing a gene or quantitative trait loci (QTL) into an elitecultivar or breeding line via backcrossing. Markers linked to the genecan be used to select plants possessing the desired trait, and markersthroughout the genome can be used to select plants that are geneticallysimilar to the recurrent parent (Young and Tanksley (1989) Theor. Appl.Genet. 77:95-101; Hospital et al. (1992) Genetics 132:1199-210).

Most of the plant fertility restorer genes have been cloned via amap-based cloning strategy. To date, five restorer genes have beenisolated from several plant species including maize (Zea Mays L.) (Cuiet al. (1996) Science 272:1334-6; Liu et al. (2001) Plant Cell13:1063-78), petunia (Petunia hybrida) (Bentolila et al. (2002) Proc.Natl. Acad. Sci. USA 99:10887-92, radish (Raphanus sativus L.) (Brown etal. (2003) Plant J. 35:262-72; Desloire et al. (2003) EMBO Rep. 4:1-7;Koizuka et al. (2003) Plant J. 34:407-15), sorghum (Sorghum bicolor L.)(Klein et al. (2005) Theor. Appl. Genet. 111:994-1012) and rice (Oryzasativa L.) (Kazama and Toriyama (2003) FEBS Lett. 544:99-102; Akagi etal. (2004) Theor. Appl. Genet. 108:1449-57; Komori et al. (2004) PlantJ. 37:315-25; Wang et al. (2006) Plant Cell 18:676-87. All of theidentified restorer genes, except for Rf2 in maize, encode differentpentatricopeptide repeat (PPR) proteins. The PPR protein contains 2 to27 repeats of 35 amino acids, referred to as PPR motifs (Small andPeeters, 2000). Many PPR proteins are targeted to mitochondria where theCMS-associated genes and products are located (Lurin et al., 2004).

Additional information regarding fertility restorer genes from maize,rice, petunia, and radish may be found in U.S. Patent Application Ser.No. US2006/0253931, and in U.S. Pat. Nos. 5,981,833; 5,624,842;4,569,152; 6,951,970; 6,392,127; 7,612,251; 7,314,971; 7,017,375;7,164,058; and 5,644,066, all of which are incorporated herein byreference.

BRIEF SUMMARY OF THE DISCLOSURE

In some embodiments, the present disclosure provides a method forselecting a plant comprising a functional restorer gene for maize S-typecytoplasmic male sterility. The method comprises the steps of (a)screening a population of plants for at least one marker nucleic acid,wherein the marker nucleic acid comprises an allele linked to thefunctional restorer gene for maize S-type cytoplasmic male sterility;(b) detecting the marker nucleic acid; (c) identifying a plantcomprising the marker nucleic acid; and (d) selecting the plantcomprising the marker nucleic acid, wherein the plant comprising themarker nucleic acid further comprises the functional restorer gene formaize S-type cytoplasmic male sterility.

In other embodiments, the present disclosure provides a method forrestoring fertility in a progeny of an S-type cytoplasmic male sterileplant. The method comprises the steps of (a) crossing a female plantwith a male plant to generate a population of progeny plants, whereinthe female plant is an S-type cytoplasmic male sterile plant, andwherein the male plant possesses a functional restorer gene for S-typecytoplasmic male sterility; (b) screening the population of progenyplants to identify a fertile progeny plant comprising at least onemarker nucleic acid comprising an allele linked to the functionalrestorer gene for maize S-type cytoplasmic male sterility; (c) selectingthe fertile progeny plant comprising at least one marker nucleic acidcomprising an allele linked to the functional restorer gene for maizeS-type cytoplasmic male sterility; and (d) propagating the fertileprogeny plant, wherein the fertile progeny plant comprises thefunctional restorer gene for maize S-type cytoplasmic male sterility.

In some embodiments, the present disclosure provides a method fortransferring an Rf3 gene into a progeny plant. The method comprises thesteps of (a) crossing a first parent plant and a second parent plant toproduce a progeny plant, wherein at least one parent plant comprises theRf3 gene; (b) analyzing the progeny plant for the presence of at leastone marker that is linked to the Rf3 gene to obtain an Rf3 progenyplant; (c) backcrossing the Rf3 progeny plant with either the firstparent plant or the second parent plant to produce a next-generationprogeny plant; and (d) analyzing the next-generation progeny plant forthe presence of the at least one marker that is linked to the Rf3 geneto obtain an Rf3 next-generation progeny plant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a QTL plot showing the LOD scores of markers associated withthe CMS-S fertility restorer QTL. FIG. 1B is a genetic map of chromosome2, including the CMS-S fertility restorer QTL.

SEQUENCE LISTING

SEQ ID NOs:1-44 show exemplary sequences of markers and primers that arelinked (e.g., linked; tightly linked; or extremely tightly linked) tothe maize Rf3 gene and are within the 8.3 Mb region on chromosome 2 thatcontains the Rf3 locus.

SEQ ID NOs:45-68 show exemplary sequences of polymorphic markers andprimers developed from several PPR genes.

SEQ ID NOs: 69-85 show exemplary sequences of polymorphic markers andprimers developed from the PPR2 gene.

SEQ ID NOs: 86-91 show exemplary sequences of primers and probes for theRf3 allele specific TaqMan® assay and the internal control, ElongationFactor α-1.

SEQ ID NO:92 is a cDNA sequence for the Mo17 Rf3-PPR2 gene (Zea mayscultivar S-Mo17(Rf3/Rf3) PPR-814a mRNA, complete cds; Sequence ID:gb|FJ176574.1).

DETAILED DESCRIPTION I. Terms

Backcrossing: Backcrossing methods may be used to introduce a nucleicacid sequence into a plant. The backcrossing technique has been widelyused for decades to introduce new traits into plants. Jensen, N., Ed.Plant Breeding Methodology, John Wiley & Sons, Inc., 1988. In a typicalbackcross protocol, the original variety of interest (recurrent parent)is crossed to a second variety (non-recurrent parent) that carries agene of interest to be transferred. The resulting progeny from thiscross are then crossed again to the recurrent parent, and the process isrepeated until a plant is obtained wherein essentially all of thedesired morphological and physiological characteristics of the recurrentplant are recovered in the converted plant, in addition to thetransferred gene from the nonrecurrent parent.

Linked, tightly linked, and extremely tightly linked: As used herein,linkage between genes or markers refers to the phenomenon in which genesor markers on a chromosome show a measurable probability of being passedon together to individuals in the next generation. The closer two genesor markers are to each other, the closer to (1) this probabilitybecomes. Thus, the term “linked” may refer to one or more genes ormarkers that are passed together with a gene with a probability greaterthan 0.5 (which is expected from independent assortment wheremarkers/genes are located on different chromosomes). Because theproximity of two genes or markers on a chromosome is directly related tothe probability that the genes or markers will be passed together toindividuals in the next generation, the term “linked” may also referherein to one or more genes or markers that are located within about 8.5Mb of one another on the same maize chromosome.

Thus, two “linked” genes or markers may be separated by about 8.5 Mb;8.3 Mb; 8.0 Mb; 7.5 Mb; 7.0 Mb; 6.5 Mb; 6.0 Mb; 5.5 Mb; 5.0 Mb; 4.5 Mb;4.0 Mb; 3.5 Mb; 3.0 Mb; 2.5 Mb; 2.0 Mb; about 1.95 Mb; about 1.9 Mb;about 1.85 Mb; about 1.8 Mb; about 1.75 Mb; about 1.7 Mb; about 1.65 Mb;about 1.6 Mb; about 1.55 Mb; about 1.5 Mb; about 1.45 Mb; about 1.4 Mb;about 1.35 Mb; about 1.3 Mb; about 1.25 Mb; about 1.2 Mb; about 1.15 Mb;about 1.1 Mb; about 1.05 Mb; about 1.0 Mb; about 0.95 Mb; about 0.9 Mb;about 0.85 Mb; about 0.8 Mb; about 0.75 Mb; about 0.7 Mb; about 0.65 Mb;about 0.6 Mb; about 0.55 Mb; about 0.5 Mb; about 0.45 Mb; about 0.4 Mb;about 0.35 Mb; about 0.3 Mb; about 0.25 Mb; about 0.2 Mb; about 0.15 Mb;about 0.1 Mb; about 0.05 Mb; about 0.025 Mb; and about 0.01 Mb.Particular examples of markers that are “linked” to Rf3 includenucleotide sequences on the long arm of chromosome 2 of the maizegenome, e.g., Mo17-14388, PZE-102180901, PZE-102180129, FG-1318,PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578,PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2,PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39.

As used herein, the term “tightly linked” may refer to one or more genesor markers that are located within about 0.5 Mb of one another on thesame maize chromosome. Thus, two “tightly linked” genes or markers maybe separated by about 0.6 Mb; about 0.55 Mb; 0.5 Mb; about 0.45 Mb;about 0.4 Mb; about 0.35 Mb; about 0.3 Mb; about 0.25 Mb; about 0.2 Mb;about 0.15 Mb; about 0.1 Mb; and about 0.05 Mb. Particular examples ofmarkers that are “tightly linked” to Rf3 include PPR1_P5_1, PPR3_P4_2,PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39.

As used herein, the term “extremely tightly linked” may refer to one ormore genes or markers that are located within about 100 kb of oneanother on the same maize chromosome. Thus, two “extremely tightlylinked” genes or markers may be separated by about 125 kb; about 120 kb;about 115 kb; about 110 kb; about 105 kb; 100 kb; about 95 kb; about 90kb; about 85 kb; about 80 kb; about 75 kb; about 70 kb; about 65 kb;about 60 kb; about 55 kb; about 50 kb; about 45 kb; about 40 kb; about35 kb; about 30 kb; about 25 kb; about 20 kb; about 15 kb; about 10 kb;about 5 kb; and about 1 kb. Particular examples of markers that are“extremely tightly linked” to Rf3 include CMSS03, CMSS10, CMSS15,CMSS34, DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39.

Linked, tightly linked, and extremely tightly genetic markers of Rf3 maybe useful in marker-assisted breeding programs to identify restorer formaize S-type cytoplasmic male sterility gene types, and to breed thistrait into maize varieties.

Locus: As used herein, the term “locus” refers to a position on thegenome that corresponds to a measurable characteristic (e.g., a trait).A SNP locus is defined by a probe that hybridizes to DNA containedwithin the locus.

Marker: As used herein, the term “marker” refers to a gene or nucleotidesequence that can be used to identify plants having a particular allele,e.g., Rf3. A marker may be described as a variation at a given genomiclocus. A genetic marker may be a short DNA sequence, such as a sequencesurrounding a single base-pair change (single nucleotide polymorphism,or “SNP”), or a long one, for example, a microsatellite/simple sequencerepeat (“SSR”). The term “marker allele” refers to the version of themarker that is present in a particular plant.

The term marker as used herein may refer to a cloned segment of maizechromosomal DNA (for example, as defined by Mo17-14388, PZE-102180901,PZE-102180129, FG-1318, PZE-102182167, PZE-102182672, PZE-102182718,PZE-102183578, PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1,PPR3_P4_2, PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15,CMSS34, DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39),and may also or alternatively refer to a DNA molecule that iscomplementary to a cloned segment of maize chromosomal DNA (for example,DNA complementary to Mo17-14388, PZE-102180901, PZE-102180129, FG-1318,PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578,PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2,PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39).

In some embodiments, the presence of a marker in a plant may be detectedthrough the use of a nucleic acid probe. A probe may be a DNA moleculeor an RNA molecule. RNA probes can be synthesized by means known in theart, for example, using a DNA molecule template. A probe may contain allor a portion of the nucleotide sequence of the marker and additional,contiguous nucleotide sequence from the maize genome. This is referredto herein as a “contiguous probe.” The additional, contiguous nucleotidesequence is referred to as “upstream” or “downstream” of the originalmarker, depending on whether the contiguous nucleotide sequence from themaize chromosome is on the 5′ or the 3′ side of the original marker, asconventionally understood. The additional, contiguous nucleotidesequence may be located between the original marker and the 8.3 Mbregion on chromosome 2 of the maize genome that is located betweenflanking markers Mo17-14388 and PZE-102184593. As is recognized by thoseof ordinary skill in the art, the process of obtaining additional,contiguous nucleotide sequence for inclusion in a marker may be repeatednearly indefinitely (limited only by the length of the chromosome),thereby identifying additional markers along the maize chromosome. Anyof the above-described markers may be used in some embodiments of thepresent disclosure.

An oligonucleotide probe sequence may be prepared synthetically or bycloning. Suitable cloning vectors are well-known to those of skill inthe art. An oligonucleotide probe may be labeled or unlabeled. A widevariety of techniques exist for labeling nucleic acid molecules,including, for example and without limitation: Radiolabeling by nicktranslation; random priming; tailing with terminal deoxytransferase; orthe like, where the nucleotides employed are labeled, for example, withradioactive ³²P. Other labels which may be used include, for example andwithout limitation: fluorophores; enzymes; enzyme substrates; enzymecofactors; enzyme inhibitors; and the like. Alternatively, the use of alabel that provides a detectable signal, by itself or in conjunctionwith other reactive agents, may be replaced by ligands to whichreceptors bind, where the receptors are labeled (for example, by theabove-indicated labels) to provide detectable signals, either bythemselves, or in conjunction with other reagents. See, e.g., Leary etal. (1983) Proc. Natl. Acad. Sci. USA 80:4045-9.

A probe may contain a nucleotide sequence that is not contiguous to thatof the original marker; this probe is referred to herein as a“noncontiguous probe.” The sequence of the noncontiguous probe islocated sufficiently close to the sequence of the original marker on themaize genome so that the noncontiguous probe is genetically linked tothe same gene (e.g., Rf3). For example, in some embodiments, anoncontiguous probe can be located within 500 kb; 450 kb; 400 kb; 350kb; 300 kb; 250 kb; 200 kb; 150 kb; 125 kb; 100 kb; 0.9 kb; 0.8 kb; 0.7kb; 0.6 kb; 0.5 kb; 0.4 kb; 0.3 kb; 0.2 kb; or 0.1 kb of the originalmarker on the maize genome.

In an embodiment, a probe may be an exact copy of a marker to bedetected. A probe may also be a nucleic acid molecule comprising, orconsisting of, a nucleotide sequence which is substantially identical toa cloned segment of maize chromosomal DNA (for example, as defined byMo17-14388, PZE-102180901, PZE-102180129, FG-1318, PZE-102182167,PZE-102182672, PZE-102182718, PZE-102183578, PZE-102183795,DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2, PPR8_P6_1, PPR3-5,PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34, DASCMS-SRf31,DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39). As used herein, the term“substantially identical” may refer to nucleotide sequences that aremore than 85% identical. For example, a substantially identicalnucleotide sequence may be 85.5%; 86%; 87%; 88%; 89%; 90%; 91%; 92%;93%; 94%; 95%; 96%; 97%; 98%; 99% or 99.5% identical to the referencesequence.

In an embodiment, a probe may also be a nucleic acid molecule that is“specifically hybridizable” or “specifically complementary” to an exactcopy of the marker to be detected (“DNA target”). “Specificallyhybridizable” and “specifically complementary” are terms that indicate asufficient degree of complementarity such that stable and specificbinding occurs between the nucleic acid molecule and the DNA target. Anucleic acid molecule need not be 100% complementary to its targetsequence to be specifically hybridizable. A nucleic acid molecule isspecifically hybridizable when there is a sufficient degree ofcomplementarity to avoid non-specific binding of the nucleic acid tonon-target sequences under conditions where specific binding is desired,for example, under stringent hybridization conditions.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ and/or Mg⁺⁺ concentration) of thehybridization buffer will determine the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are known to those of ordinary skill in the art, and arediscussed, for example, in Sambrook et al. (ed.) Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames andHiggins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985.Further detailed instruction and guidance with regard to thehybridization of nucleic acids may be found, for example, in Tijssen,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” in Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, Part I,Chapter 2, Elsevier, N.Y., 1993; and Ausubel et al., Eds., CurrentProtocols in Molecular Biology, Chapter 2, Greene Publishing andWiley-Interscience, NY, 1995.

As used herein, the term “stringent conditions” encompasses conditionsunder which hybridization will only occur if there is less than 25%mismatch between the hybridization molecule and the DNA target.Stringent conditions include further particular levels of stringency.Thus, as used herein, “moderate stringency” conditions are those underwhich molecules with more than 25% sequence mismatch will not hybridize;conditions of “medium stringency” are those under which molecules withmore than 15% mismatch will not hybridize; and conditions of “highstringency” are those under which sequences with more than 10% mismatchwill not hybridize. Conditions of “very high stringency” are those underwhich sequences with more than 6% mismatch will not hybridize.

In particular embodiments, stringent conditions include hybridization at65° C. in 6× saline-sodium citrate (SSC) buffer, 5×Denhardt's solution,0.5% SDS, and 100 μg sheared salmon testes DNA, followed by 15-30 minutesequential washes at 65° C. in 2×SSC buffer and 0.5% SDS, followed by1×SSC buffer and 0.5% SDS, and finally 0.2×SSC buffer and 0.5% SDS.

With respect to all probes discussed, supra, the probe may compriseadditional nucleic acid sequences, for example: gDNA; promoters;transcription signals; and/or vector sequences. Any of the probesdiscussed, supra, may be used to define additional markers that arelinked to a gene involved in restoring fertility to S-type cytoplasmicsterile maize (e.g., Rf3). Markers thus identified may be equivalent toexemplary markers named in the present disclosure and, thus, are withinthe scope of the present disclosure.

Marker-assisted breeding: As used herein, the term “marker-assistedbreeding” may refer to an approach to breeding directly for one or morecomplex traits (e.g., CMS-S restorer of fertility). In practice, plantbreeders attempt to identify readily detectable traits, such as flowercolor, seed coat appearance, or isozyme variants that are linked to anagronomically desired trait. The plant breeders then follow theagronomic trait in the segregating, breeding populations by followingthe segregation of the easily detectable trait. However, there are veryfew of these linkage relationships available for use in plant breeding.

Marker-assisted breeding provides a time- and cost-efficient process forimprovement of plant varieties. Several examples of the application ofmarker-assisted breeding involve the use of isozyme markers. See, e.g.,Tanksley and Orton, eds. (1983) Isozymes in Plant Breeding and Genetics,Amsterdam: Elsevier. One example is an isozyme marker associated with agene for resistance to a nematode pest in tomato. The resistance,controlled by a gene designated Mi, is located on chromosome 6 of tomatoand is very tightly linked to Aps1, an acid phosphatase isozyme. Use ofthe Aps1 isozyme marker to indirectly select for the Mi gene providedthe advantages that segregation in a population can be determinedunequivocally with standard electrophoretic techniques; the isozymemarker can be scored in seedling tissue, eliminating the need tomaintain plants to maturity; and co-dominance of the isozyme markeralleles allows discrimination between homozygotes and heterozygotes. SeeRick (1983) in Tanksley and Orton, supra.

Operably linked: A first nucleotide sequence is “operably linked” with asecond nucleic acid sequence when the first nucleic acid sequence is ina functional relationship with the second nucleic acid sequence. Forexample, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.When recombinantly produced, operably linked nucleic acid sequences aregenerally contiguous, and, where necessary to join two protein-codingregions, in the same reading frame (e.g., in a polycistronic ORF).However, nucleic acids need not be contiguous to be operably linked.

Promoter: As used herein, the term “promoter” refers to a region of DNAthat may be upstream from the start of transcription, and that may beinvolved in recognition and binding of RNA polymerase and other proteinsto initiate transcription. A promoter may be operably linked to a genefor expression in a cell, or a promoter may be operably linked to anucleotide sequence encoding a signal sequence which may be operablylinked to a gene for expression in a cell. A “plant promoter” may be apromoter capable of initiating transcription in plant cells. Examples ofpromoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma.Such promoters are referred to as “tissue-preferred.” Promoters whichinitiate transcription only in certain tissues are referred to as“tissue-specific.” A “cell type-specific” promoter primarily drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” promoter may be apromoter which may be under environmental control. Examples ofenvironmental conditions that may initiate transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which may be active under mostenvironmental conditions.

Any inducible promoter can be used in some embodiments of the presentdisclosure. See Ward et al. (1993) Plant Mol. Biol. 22:361-366. With aninducible promoter, the rate of transcription increases in response toan inducing agent. Exemplary inducible promoters include, but are notlimited to: Promoters from the ACEI system that responds to copper; 1n2gene from maize that responds to benzenesulfonamide herbicide safeners;Tet repressor from Tn10; and the inducible promoter from a steroidhormone gene, the transcriptional activity of which may be induced by aglucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci.USA 88:0421).

Exemplary constitutive promoters include, but are not limited to:promoters from plant viruses, such as the 35S promoter from CaMV;promoters from rice actin genes; ubiquitin promoters; pEMU; MAS; maizeH3 histone promoter; and the ALS promoter, Xba1/NcoI fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xba1/NcoI fragment) (see, e.g., WO 96/30530).

Any tissue-specific or tissue-preferred promoter may also be utilized insome embodiments the present disclosure. Plants transformed with a geneoperably linked to a tissue-specific promoter may produce the proteinproduct of the transgene exclusively, or preferentially, in a specifictissue. Exemplary tissue-specific or tissue-preferred promoters include,but are not limited to: a root-preferred promoter, such as that from thephaseolin gene; a leaf-specific and light-induced promoter such as thatfrom cab or rubisco; an anther-specific promoter such as that fromLAT52; a pollen-specific promoter such as that from Zm13; and amicrospore-preferred promoter such as that from apg.

Sequence identity: The terms “sequence identity” or “identity,” as usedherein in the context of two nucleic acid or polypeptide sequences, mayrefer to the residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window.

When percentage of sequence identity is used in reference to proteins,it is recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge, hydrophobicity, or steric effects),and therefore do not change the functional properties of the molecule.

Therefore, when sequences differ by conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution at the site of the non-identicalresidue. Sequences that differ by such conservative substitutions aresaid to have “sequence similarity” or “similarity.” Techniques formaking this adjustment are well known to those of ordinary skill in theart. Typically, such techniques involve scoring a conservativesubstitution as a partial, rather than a full, mismatch, therebyincreasing the percentage sequence identity. For example, where anidentical amino acid is given a score between 0 and 1, and anon-conservative substitution is given a score of 0, a conservativesubstitution is given a score between 0 and 1. The scoring ofconservative substitutions may be calculated, for example, asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

As used herein, the term “percentage of sequence identity” may refer tothe value determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleotideor amino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity.

Single-nucleotide polymorphism (SNP): As used herein, the term“single-nucleotide polymorphism” may refer to a DNA sequence variationoccurring when a single nucleotide in the genome (or other sharedsequence) differs between members of a species or paired chromosomes inan individual.

Within a population, SNPs can be assigned a minor allele frequency thelowest allele frequency at a locus that is observed in a particularpopulation. This is simply the lesser of the two allele frequencies forsingle-nucleotide polymorphisms. There are variations between humanpopulations, so a SNP allele that is common in one geographical orethnic group may be much rarer in another.

Single nucleotide polymorphisms may fall within coding sequences ofgenes, non-coding regions of genes, or in the intergenic regions betweengenes. SNPs within a coding sequence will not necessarily change theamino acid sequence of the protein that is produced, due to degeneracyof the genetic code. A SNP in which both forms lead to the samepolypeptide sequence is termed “synonymous” (sometimes referred to asilent mutation). If a different polypeptide sequence is produced, theyare termed “non-synonymous.” A non-synonymous change may either bemissense or nonsense, where a missense change results in a differentamino acid and a nonsense change results in a premature stop codon. SNPsthat are not in protein-coding regions may still have consequences forgene splicing, transcription factor binding, or the sequence ofnon-coding RNA. SNPs are usually biallelic and thus easily assayed inplants and animals. Sachidanandam (2001) Nature 409:928-33.

Trait or phenotype: The terms “trait” and “phenotype” are usedinterchangeably herein. For the purposes of the present disclosure, atrait of particular interest is fertility restoration of S-type CMS.

II. The Rf3 Gene and Molecular Markers Thereof

The present disclosure provides particular embodiments of a geneimpacting male fertility in plants, maize Rf3, and linked geneticmarkers thereof, which can be useful in a variety of systems to controlmale fertility. Furthermore, the polymorphism inherent in the disclosedlinked genetic markers allows a plant breeder to follow the particularallele of the gene, Rf3 or rf3, in a segregating population.

The Rf3 gene was initially mapped to chromosome 2 in an F2 population of450 individuals derived from a cross of a male sterile line ‘4XP811-D’with a restoring line ‘LH60.’ In view of the practical importance ofcytoplasmic male sterility and pollen fertility restoration in maizehybrid seed production, and of the necessity of cytoplasm sourcediversification, fine mapping of the maize Rf3 restorer gene for CMS-Sto a very small region by using molecular markers with a KASPar™genotyping technique and the identification of the maize Rf3 genethrough map-based cloning are described. It was determined that Rf3 is asingle dominant restorer gene for CMS-S in two maize inbreds: LH60 andMBB56.

The Rf3 gene encodes a pentatricopeptide repeat (PPR) protein, as donearly all other fertility restorer genes. The identification of the Rf3gene and Rf3 gene markers may greatly facilitate the development anddeployment of the CMS-S fertility restoration trait broadly in plantgermplasm. In some embodiments, markers that are linked to (e.g.,linked; tightly linked; or extremely tightly linked) or reside withinthe maize Rf3 gene, or the maize Rf3 gene sequence itself, may be usedto introduce the maize Rf3 gene into plant organisms.

Rf3 was mapped using SNP markers to a region of approximately 8.3 Mbinterval by flanked markers Mo17-14388 and DAS-PZ-13844 on the long armof chromosome 2. Annotation of this interval with reference genomesequence from Zea mays c.v. B73 revealed 10 PPR genes and furtherfine-mapped Rf3 gene to a small interval of approximately 1.3 Mb.

In the present disclosure, molecular markers that are linked to themaize CMS-S restorer gene, Rf3, are provided. DNA segments containingsequences involved in restoration of fertility to CMS-S plants areidentified. These segments are located between markers that are linkedto the Rf3 gene (SEQ ID NO:92). Nucleic acid molecules comprising theRf3 gene are also provided. The segments identified, and the markersthereof, are described herein, in part, by their position in aparticular region on the long arm of maize chromosome 2.

The position of the segments identified, and the markers thereof, whenexpressed as recombination frequencies or map units, are provided hereinas a matter of general information. The embodiments described hereinwere obtained using a maize population, 4XP811-DxLH60. However, thepositions of particular segments and markers as map units are expressedwith reference to the publically available B73 maize inbred genomesequence (B73 RefGen v2), which may be found at Maize GDB. It isexpected that numbers given for particular segments and markers as mapunits may vary from cultivar to cultivar and are not part of theessential definition of the DNA segments and markers, which DNA segmentsand markers are otherwise described, for example, by nucleotidesequence.

The dominant allele of the Rf3 gene controls fertility restoration inthe CMS-S/Rf3 system. In particular embodiments, an Rf3 gene is provided(SEQ ID NO:92). In some embodiments, the present disclosure alsoincludes those nucleotide sequences which are substantially identical tothe Rf3 sequence (SEQ ID NO:92). For example, in some embodiments, anucleic acid molecule is an Rf3 homologue that is at least about 85%identical to the Rf3 sequence (SEQ ID NO:92). An Rf3 homologue may be86%; 87%; 88%; 89%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99% or99.5% identical to the Rf3 sequence (SEQ ID NO:92). Such an Rf3homologue may be readily identified and isolated from any complete orpartial genomes readily available to those of skill in the art for avariety of organisms.

Some embodiments also include functional variants of the Rf3 gene.Functional variants of Rf3 include, for example, the Rf3 sequence (SEQID NO:92) comprising one or more nucleotide substitutions, deletions, orinsertions, wherein the functional variant restores male fertility toCMS-S corn, as may be measured by routine techniques well-known to thoseof ordinary skill in the art. For example, the capability of aparticular variant of the Rf3 gene to restore male fertility to CMS-Scorn may be determined by routine introduction of the mutation orfragment into plants homozygous for a sterile rf3 allele, followed byroutine observation of the plant for male sterility. Functional variantsof the Rf3 gene may be created by site-directed mutagenesis, inducedmutation, or they may occur as allelic variants (polymorphisms, e.g.,SNPs).

In some embodiments, therefore, functional variants of the Rf3 gene maybe mutations of Rf3, or fragments smaller than entire sequence of Rf3,which retain the male sterility controlling properties of the Rf3 gene.Such mutations and fragments are therefore considered to be within thescope of the subject disclosure. One of ordinary skill in the art canreadily determine whether a mutation or fragment of the Rf3 sequence setforth herein retains the properties of the Rf3 gene.

III. Methods of Using the Rf3 Gene

The Rf3 gene described herein may be used via techniques known by one ofskill in the art to manipulate a gene to cause a desired effect. Forexample and without limitation, the Rf3 gene may be used: to introduce amutant Rf3 sequence into a plant to cause sterility; to introduce amutation into the native Rf3 sequence; to introduce an antisense nucleicacid molecule targeting Rf3 DNA or RNA into a plant to affect fertility;to use hairpin formations; or to link Rf3 sequence(s) with other nucleicacid sequences to control the expression of Rf3 gene product. Forexample, in some embodiments, the Rf3 gene (SEQ ID NO:92) may be used tofacilitate the utilization of the CMS-S/Rf3 male fertility system inconjunction with other genes or mutants impacting male fertility inmaize.

In some embodiments, the Rf3 gene may be introduced into a maize plantthat is suitable for use in a male fertility system other than theCMS-S/Rf3 male fertility system. Alternatively, a gene or mutant geneother than Rf3 may be introduced into a maize plant that is suitable foruse in the CMS-S/Rf3 male fertility system, such that the introducedgene or mutant gene may be used to provide additional or complementaryfertility control. Specific examples of other male fertility genes andmutations in maize include: CMS-T/Rf1; CMS-T/Rf2; CMS-S/Rf3; ms1(Singleton and Jones (1930) J. Hered. 21:266-8); ms2 and ms3 (Eyster(1931) J. Hered. 22:99-102); mss, ms7, ms8, ms9, ms10, ms1 1, ms12,ms13, and ms14 (Beadle (1932) Genetics 17:413-31); ms17 (Emerson (1932)Science 75:566); ms20 (Eyster (1934) Bibliographia Genetica 11:187-392);ms23 and ms24 (West and Albertsen (1985) MNL 59:87); ms25 and ms26(Loukides et al. (1995) Am. J. Bot. 82:1017-23); ms27 and ms38(Albertsen et al. (1996) MNL 70:30-1); ms28 (Golubovskaya (1979) MNL53:66-70); ms29 and ms31 (Trimnell et al. (1998) MNL 72:37-38); ms30(Albertsen et al. (1999) MNL 73:48); ms32, ms36, and ms37 (Trimnell etal. (1999) MNL 73:48-50); ms33 and ms34 (Patterson (1995) MNL 69:126-8);ms43 (Golubovskaya (1979) Int. Rev. Cytol. 58:247-90); ms45 (Albertsenet al. (1993) Proc. Annu. Corn Sorghum Ind. Res. Conf. 48:224-33; andms48, ms49, and ms50 (Trimnell et al. (2002) MNL 76:38-9).

When a nucleic acid sequence (e.g., Rf3) is “introduced” into anorganism, such as a plant, the technique or methodology used for theintroduction of a nucleic acid molecule comprising the particularsequence is not essential to the subject disclosure, and may occur byany technique or methodology known to those of skill in the art. Forexample, a nucleic acid molecule may be introduced by directtransformation methods, such as Agrobacterium-mediated transformation ofplant tissue; microprojectile bombardment; electroporation; etc.Alternatively, a nucleic acid molecule may be introduced by crossing aplant having the particular nucleotide sequence with another plant, suchthat progeny have the nucleotide sequence incorporated into theirgenome. Such breeding techniques are well-known to one skilled in theart. Marker-assisted breeding techniques, as disclosed herein, maygreatly facilitate the incorporation of Rf3 through such crosses.

In embodiments wherein the Rf3 gene is introduced to an organism, it maybe desirable for the Rf3 gene to be introduced in such a manner that theRf3 gene is operably linked to one or more regulatory sequences, forexample, introduction via the use of a plasmid comprising the Rf3 geneoperably linked to the desired regulatory sequences. Regulatorysequences useful in the expression of heterologous nucleic acidsequences are well-known in the art, and include, for example andwithout limitation: Promoters (e.g., constitutive promoters;tissue-specific promoters; and developmental stage-specific promoters);termination sequences; enhancer sequences; subcellular targetingsequences; stabilizing or leader sequences; and introns.

In some embodiments, the Rf3 gene may be introduced to an organism withone or more additional desirable nucleic acid sequences (for example,genes). Additional desirable nucleic acid sequences may include, forexample: Genes encoding foreign proteins; agronomic genes; plant diseaseresistance genes; genes conferring resistance to a plant pest; genesconferring resistance to an herbicide; and genes that confer orcontribute to a value-added trait (e.g., modified fatty acid metabolism;decreased phytate content; and modified carbohydrate composition).Examples of all the aforementioned nucleic acid sequences are known tothose of skill in the art.

The Rf3 gene may also be introduced to an organism with one or moremarker genes operably linked to a regulatory element (a promoter, forexample) that allows transformed cells containing the marker to beeither recovered by negative selection (i.e., inhibiting growth of cellsthat do not contain the selectable marker gene) or by positive selection(i.e., screening for the product encoded by the genetic marker). Manyselectable marker genes for transformation are well known in thetransformation arts and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich may be insensitive to the inhibitor. Positive selection methodsare also known in the art. Examples of marker genes suitable for use inplant cells may include, for example, and without limitation: Theneomycin phosphotransferase II (nptII) gene (Fraley et al. (1983) Proc.Natl. Acad. Sci. USA 80:4803); the hygromycin phosphotransferase gene(Vanden Elzen et al. (1985) Plant Mol. Biol. 5:299); gentamycin acetyltransferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyltransferase, and the bleomycin resistance determinant (See, e.g.,Hayford et al. (1988) Plant Physiol. 86:1216; Jones et al. (1987) Mol.Gen. Genet. 210:86); Svab et al. (1990) Plant Mol. Biol. 14:197; andHille et al. (1986) Plant Mol. Biol. 7:171); selectable marker genesthat confer resistance to herbicides, such as glyphosate, glufosinate orbromoxynil (See, e.g., Comai et al. (1985) Nature 317:741-744;Gordon-Kamm et al. (1990) Plant Cell 2:603-618; and Stalker et al.(1988) Science 242:419-423); mouse dihydrofolate reductase (Eichholtz etal. (1987) Somatic Cell Mol. Genet. 13:67); plant5-enolpyruvylshikimate-3-phosphate synthase (Shah et al. (1986) Science233:478); plant acetolactate synthase (Charest et al. (1990) Plant CellRep. 8:643).

Another class of marker genes suitable for plant transformation employsscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance, such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues, and are frequently referred to as “reporter genes,”because they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningtransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase and chloramphenicol acetyltransferase. See, e.g., Jefferson(1987) Plant Mol. Biol. Rep. 5:387; Teeri et al. (1989) EMBO J. 8:343;Koncz et al. (1987) Proc. Natl. Acad. Sci U.S.A. 84:131; and DeBlock etal. (1984) EMBO J. 3:1681.

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green™, p. 1-4, 1993; and Naleway etal. (1991) J. Cell Biol. 115:151a. Further, genes encoding FluorescentProteins (e.g., GFP, EGFP, EBFP, ECFP, and YFP) have been utilized asmarkers for gene expression in prokaryotic and eukaryotic cells. SeeChalfie et al. (1994) Science 263:802. Fluorescent proteins andmutations of fluorescent proteins may be used as screenable markers.

In some embodiments, the maize Rf3 gene and fragments or segments of themaize Rf3 gene disclosed herein may be used to identify homologous Rf3gene sequences from organisms other than maize (e.g., by sequencecomparison). Sequences from organisms other than maize that arehomologous to the maize Rf3 gene may be identified and isolatedaccording to well-known techniques, for example, based on their sequencehomology to the Rf3 sequence. For example, all or part of the Rf3 codingsequence may be used as a probe which specifically hybridizes to othersequences present in a population of cloned genomic DNA fragments (i.e.,a genomic library) from an organism according to routine techniques.Thus, in some embodiments, the present disclosure includes thosenucleotide sequences which specifically hybridize to the Rf3 sequence.

Alternatively, sequences from organisms other than maize that arehomologous to the maize Rf3 gene may be identified and isolated bysequence comparison. For example, the complete or partial sequencedgenome of an organism may be searched according to routine techniqueswith the maize Rf3 to identify genes within the genome of the organismthat share a high degree of sequence identity with maize Rf3, and aretherefore likely Rf3 homologues.

For example, all or part of the maize Rf3 sequence may be used as a“reference sequence.” Generally, nucleic acid sequences (e.g., cloned orgenomic DNA fragments of a genomic library) that are compared to thereference sequence comprise a “comparison window,” which is a specificcontiguous segment of the nucleic acid sequence. The comparison windowmay comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The comparison window istypically at least 20 contiguous nucleotides in length, but may be 30,40, 50, 100, or 200 nucleotides in length, or longer. To avoid a highsimilarity to the reference sequence due to inclusion of deletions inthe polynucleotide sequence comparison window, a “gap penalty” may beintroduced to be subtracted from the number of nucleotide matches.

Methods of aligning sequences for comparison are well-known in the art.The determination of percent sequence identity between any two sequencescan be accomplished using available mathematical algorithms.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988), CABIOS 4:11-7; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970), J. Mol. Biol. 48:443-53; thesearch-for-local-alignment method of Pearson and Lipman (1988), Proc.Natl. Acad. Sci. USA 85:2444-8; the algorithm of Karlin and Altschul(1990), Proc. Natl. Acad. Sci. USA 87:2264, and Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-7.

One of ordinary skill in the art can implement these mathematicalalgorithms on a computer for comparison of sequences to determinesequence identity, or to search a database comprising a plurality ofsequences (e.g., an organism genome database) according to sharedsequence identity with a reference sequence. Such implementationsinclude, but are not limited to, CLUSTAL in the PC/Gene program(Intelligenetics, Mountain View, Calif.); and the ALIGN program and GAP,BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics SoftwarePackage, v. 10 (Accelrys Inc., San Diego, Calif.). Sequence alignmentsusing these programs may be performed using their default parameters.Alternatively, it may be desirable to modify the default parameters insome searches (e.g., altering the value of a gap penalty). The selectionof a particular computer implementation of mathematical algorithms forcalculation of sequence identity, and the selection of parameter valuesfor use in a selected algorithm, are within the discretion of one ofskill in the art.

IV. Methods of Using Rf3 Molecular Markers

Methods of using nucleic acid molecular markers that are linked to orthat reside within the Rf3 gene to identify plants with a functionalrestorer gene for S-type CMS may result in a cost savings for plantdevelopers, because such methods may eliminate the need to cross plantscomprising a functional restorer gene with CMS plant lines and thenphenotype the progenies of the cross.

Additional markers can be identified as equivalent to any of theexemplary markers named herein (e.g., Mo17-14388, PZE-102180901,PZE-102180129, FG-1318, PZE-102182167, PZE-102182672, PZE-102182718,PZE-102183578, PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1,PPR3_P4_2, PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15,CMSS34, DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39),for example, by determining the frequency of recombination between theadditional marker and an exemplary named marker. Such determinations mayutilize an improved method of orthogonal contrasts based on the methodof Mather (1931), The Measurement of Linkage in Heredity, Methuen & Co.,London, followed by a test of maximum likelihood to determine arecombination frequency. Allard (1956) Hilgardia 24:235-78. If the valueof the recombination frequency is less than or equal to 0.10 (i.e., 10%)in any maize cultivar, then the additional marker is consideredequivalent to the particular reference marker for the purposes of use inthe presently disclosed methods.

A means for restoring fertility to CMS-S corn may include a nucleic acidsequence from a plant, the detection of said nucleic acid provides astrong indication that the plant comprising the nucleic acid sequencecomprises a functional restorer of CMS-S gene. In some examples, a meansfor restoring fertility to CMS-S corn is a marker that is linked to(e.g., linked; tightly linked; or extremely tightly linked) or thatresides within the Rf3 gene.

A means for identifying corn plants carrying a gene for restoringfertility to CMS-S corn may be a molecule that presents a detectablesignal when added to a sample obtained from a plant carrying a gene forrestoring fertility to CMS-S corn. Specific hybridization of nucleicacids is a detectable signal, and a nucleic acid probe that specificallyhybridizes to a CMS-S restorer gene, or a different genomic nucleic acidsequence that is an indicator of the presence of a functional CMS-Srestorer gene, may therefore be a means for identifying corn plantscarrying a gene for restoring fertility to CMS-S corn. In some examples,a means for identifying plants carrying a gene for restoring fertilityto CMS-S corn is a probe that specifically hybridizes to a marker thatis linked to (e.g., linked; tightly linked; or extremely tightly linked)or that resides within the maize Rf3 gene.

In some embodiments, markers flanking the Rf3 gene may be used totransfer segment(s) of donor parent DNA that unequivocally contain theRf3 gene. In particular embodiments, the markers are selected from thegroup of markers comprising Mo17-14388, PZE-102180901, PZE-102180129,FG-1318, PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578,PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2,PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39. In otherembodiments, equivalent markers are selected from the group of markerscomprising Mo17-14388, PZE-102180901, PZE-102180129, FG-1318,PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578,PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2,PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39. In someembodiments, a method for using markers flanking the Rf3 gene totransfer segment(s) of donor parent DNA that contain the Rf3 gene maycomprise analyzing the genomic DNA of two parent plants with probes thatare specifically hybridizable to markers linked (e.g., linked; tightlylinked; or extremely tightly linked) to the Rf3 gene; sexually crossingthe two parental plant genotypes to obtain a progeny population, andanalyzing those progeny for the presence of the markers linked (e.g.,linked; tightly linked; or extremely tightly linked) to the Rf3 gene;backcrossing the progeny that contain the markers linked (e.g., linked;tightly linked; or extremely tightly linked) to the Rf3 gene to therecipient genotype to produce a first backcross population, and thencontinuing with a backcrossing program until a final progeny is obtainedthat comprises any desired trait(s) exhibited by the parent genotype andthe Rf3 gene. In particular embodiments, individual progeny obtained ineach crossing and backcrossing step are selected by Rf3 marker analysisat each generation. In some embodiments, analysis of the genomic DNA ofthe two parent plants with probes that are specifically hybridizable tomarkers linked (e.g., linked; tightly linked; or extremely tightlylinked) to the Rf3 gene reveals that one of the parent plants comprisesfewer of the linked markers to which the probes specifically hybridize,or none of the linked markers to which the probes specificallyhybridize.

In some embodiments, markers that are linked to (e.g., linked; tightlylinked; or extremely tightly linked) or that reside within the maize Rf3gene, or the maize Rf3 gene sequence itself, may be used to introducethe maize Rf3 gene into a maize plant by genetic transformation. Inparticular embodiments, the markers are selected from the group ofmarkers comprising Mo17-14388, PZE-102180901, PZE-102180129, FG-1318,PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578,PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2,PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39. In otherembodiments, equivalent markers are selected from the group of markerscomprising Mo17-14388, PZE-102180901, PZE-102180129, FG-1318,PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578,PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2,PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39. In someembodiments, a method for introducing the maize Rf3 gene into a maizeplant by genetic recombination may comprise analyzing the genomic DNA ofa plant (e.g., a maize plant) with probes that are specificallyhybridizable to markers linked (e.g., linked; tightly linked; orextremely tightly linked) to the Rf3 gene or the Rf3 gene itself toidentify the Rf3 gene in the plant; isolating a segment of the genomicDNA of the plant comprising the Rf3 gene, for example, by extracting thegenomic DNA and digesting the genomic DNA with one or more restrictionendonuclease enzymes; optionally amplifying the isolated segment of DNA;introducing the isolated segment of DNA into a cell or tissue of a hostmaize plant; and analyzing the DNA of the host maize plant with probesthat are specifically hybridizable to markers linked (e.g., linked;tightly linked; or extremely tightly linked) to the Rf3 gene or the Rf3gene itself to identify the Rf3 gene in the host maize plant. Inparticular embodiments, the isolated segment of DNA may be introducedinto the host maize plant such that it is stably integrated into thegenome of the host maize plant.

In some embodiments, markers that are linked to (e.g., linked; tightlylinked; or extremely tightly linked) or that reside within the maize Rf3gene, or the maize Rf3 gene sequence itself, may be used to introducethe maize Rf3 gene into other organisms, for example, plants. Inparticular embodiments, the markers are selected from the group ofmarkers comprising Mo17-14388, PZE-102180901, PZE-102180129, FG-1318,PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578,PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2,PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39. In otherembodiments, equivalent markers are selected from the group of markerscomprising Mo17-14388, PZE-102180901, PZE-102180129, FG-1318,PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578,PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1, PPR3_P4_2,PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, and DASCMS-SRf39. In someembodiments, a method for introducing the maize Rf3 gene into anorganism other than maize may comprise analyzing the genomic DNA of aplant (e.g., a maize plant) with probes that are specificallyhybridizable to markers linked (e.g., linked; tightly linked; orextremely tightly linked) to the Rf3 gene or the Rf3 gene itself toidentify the Rf3 gene in the plant; isolating a segment of the genomicDNA of the plant comprising the Rf3 gene, for example, by extracting thegenomic DNA and digesting the genomic DNA with one or more restrictionendonuclease enzymes; optionally amplifying the isolated segment of DNA;introducing the isolated segment of DNA into an organism other thanmaize; and analyzing the DNA of the organism other than maize withprobes that are specifically hybridizable to markers linked (e.g.,linked; tightly linked; or extremely tightly linked) to the Rf3 gene orthe Rf3 gene itself to identify the Rf3 gene in the organism. Inparticular embodiments, the isolated segment of DNA may be introducedinto the organism such that it is stably integrated into the genome ofthe organism.

In some embodiments, markers that are linked to (e.g., linked; tightlylinked; or extremely tightly linked) or that reside within the Rf3 gene,or the Rf3 gene sequence itself, may be used to identify a plant with afunctional restorer gene for CMS-S male sterility. In particularembodiments, the plant is a maize plant. In some embodiments, nucleicacid molecules (e.g., genomic DNA or mRNA) may be extracted from aplant. The extracted nucleic acid molecules may then be contacted withone or more probes that are specifically hybridizable to markers linked(e.g., linked; tightly linked; or extremely tightly linked) to the Rf3gene or the Rf3 gene itself. Specific hybridization of the one or moreprobes to the extracted nucleic acid molecules is indicative of thepresence of a functional restorer gene for CMS-S male sterility in theplant.

V. Organisms Comprising the Rf3 Gene

Some embodiments of the present disclosure also provide an organismincluding a nucleic acid molecule comprising the Rf3 sequence (SEQ IDNO:92), a nucleic acid sequence that is specifically hybridizable to theRf3 sequence (SEQ ID NO:92), or a functional variant of the Rf3 sequence(SEQ ID NO:92). A suitable organism can be any suitable plant, yeast, orbacterium. By way of non-limiting example, a plant comprising theaforementioned sequences may be a plant of agronomic value, for exampleand without limitation: maize; soybean; alfalfa; wheat; rapeseed; rice;sorghum; beet; various vegetables including cucumber, tomato, peppers,etc.; various trees including apple, pear, peach, cherry, redwood, pine,oak, etc.; and various ornamental plants. In particular embodiments, theorganism may be a sexually-reproducing plant. A seed-bearing plant thatcomprises a particular nucleic acid sequence may produce seeds thatcomprise the nucleic acid sequence.

Plant cells comprising the Rf3 sequence (SEQ ID NO:92), a nucleic acidsequence that is specifically hybridizable to the Rf3 sequence (SEQ IDNO:92), or a functional variant of the Rf3 sequence (SEQ ID NO:92), maybe cultured and kept as plant tissue culture cells, or certain planthormones known in the art can be added to the culture media, therebycausing the plant tissue culture cells to differentiate and form a newplant variety, which new plant variety may be fertile or sterile. Suchplant culturing methods useful in these and other embodiments areroutine and well-known in the art.

Some embodiments of the present disclosure provide a virus (e.g., abacteriophage, or plant virus) comprising the Rf3 sequence (SEQ IDNO:92), a nucleic acid sequence that is specifically hybridizable to theRf3 sequence (SEQ ID NO:92), or a functional variant of the Rf3 sequence(SEQ ID NO:92).

VI. Various Embodiments of the Present Disclosure

In one embodiment, a method for selecting a plant comprising afunctional restorer gene for maize S-type cytoplasmic male sterility isprovided. The method comprises the steps of (a) screening a populationof plants for at least one marker nucleic acid, wherein the markernucleic acid comprises an allele linked to the functional restorer genefor maize S-type cytoplasmic male sterility; (b) detecting the markernucleic acid; (c) identifying a plant comprising the marker nucleicacid; and (d) selecting the plant comprising the marker nucleic acid,wherein the plant comprising the marker nucleic acid further comprisesthe functional restorer gene for maize S-type cytoplasmic malesterility. As used herein, the term “marker nucleic acid” means anucleic acid molecule that is utilized to determine an attribute orfeature (e.g., presence or absence, location, correlation, etc.) of amolecule, cell, or tissue.

In some embodiments, the marker nucleic acid comprises a haplotype ofalleles linked to the functional restorer gene for maize S-typecytoplasmic male sterility. In other embodiments, the marker nucleicacid is selected from the group consisting of Mo17-14388, PZE-102180901,PZE-102180129, FG-1318, PZE-102182167, PZE-102182672, PZE-102182718,PZE-102183578, PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1,PPR3_P4_2, PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15,CMSS34, DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, DASCMS-SRf39, and anycombination thereof. In yet other embodiments, the marker nucleic acidis selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, SEQ ID NO:85 and any combination thereof.

In some embodiments, the marker nucleic acid is selected from the groupconsisting of PPR1_P5_1, PPR3_P4_2, PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9,CMSS03, CMSS10, CMSS15, CMSS34, DASCMS-SRf31, DASCMS-SRf34,DASCMS-SRf321, DASCMS-SRf39, and any combination thereof. In otherembodiments, the marker nucleic acid is selected from the groupconsisting of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and anycombination thereof. In yet other embodiments, the marker nucleic acidis selected from the group consisting of CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, DASCMS-SRf39, and anycombination thereof. In other embodiments, the marker nucleic acid isselected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and anycombination thereof.

In various embodiments, at least one of the markers is associated within8.3 Mb of the functional restorer gene for maize S-type cytoplasmic malesterility. In other various embodiments, at least one of the markers isassociated within 0.5 Mb of the functional restorer gene for maizeS-type cytoplasmic male sterility. In yet other various embodiments, atleast one of the markers is associated within 100 kb of the functionalrestorer gene for maize S-type cytoplasmic male sterility.

In some embodiments, the plant is a maize plant. In another embodiment,the maize plant belongs to the Stiff Stalk heterotic group. Maizehybrids, such as temperate maize hybrids, can be derived from aheterotic group such as the Iowa Stiff Stalk heterotic group. The StiffStalk heterotic group is well known in the art of plant breeding. In yetanother embodiment, the functional restorer gene for maize S-typecytoplasmic male sterility is the Rf3 gene (SEQ ID NO:92). In anotheraspect of the present disclosure, a maize plant is obtained by thedescribed method.

In yet another aspect of the present disclosure, the method furthercomprises the steps of (e) obtaining the plant comprising the functionalrestorer gene for maize S-type cytoplasmic male sterility; (f) crossingthe plant comprising the functional restorer gene for maize S-typecytoplasmic male sterility to a second plant to produce one or moreprogeny plants; (g) evaluating the progeny plant for at least one markernucleic acid comprising an allele linked to the functional restorer genefor maize S-type cytoplasmic male sterility; and (h) selecting theprogeny plant comprising the at least one marker nucleic acid comprisingan allele linked to the functional restorer gene for maize S-typecytoplasmic male sterility.

In one embodiment, a method for restoring fertility in a progeny of anS-type cytoplasmic male sterile plant is provided. The method comprisesthe steps of (a) crossing a female plant with a male plant to generate apopulation of progeny plants, wherein the female plant is an S-typecytoplasmic male sterile plant, and wherein the male plant possesses afunctional restorer gene for S-type cytoplasmic male sterility; (b)screening the population of progeny plants to identify a fertile progenyplant comprising at least one marker nucleic acid comprising an allelelinked to the functional restorer gene for maize S-type cytoplasmic malesterility; (c) selecting the fertile progeny plant comprising at leastone marker nucleic acid comprising an allele linked to the functionalrestorer gene for maize S-type cytoplasmic male sterility; and (d)propagating the fertile progeny plant, wherein the fertile progeny plantcomprises the functional restorer gene for maize S-type cytoplasmic malesterility.

In some embodiments, the marker nucleic acid comprises a haplotype ofalleles linked to the functional restorer gene for maize S-typecytoplasmic male sterility. In other embodiments, the marker nucleicacid is selected from the group consisting of Mo17-14388, PZE-102180901,PZE-102180129, FG-1318, PZE-102182167, PZE-102182672, PZE-102182718,PZE-102183578, PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1,PPR3_P4_2, PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15,CMSS34, DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, DASCMS-SRf39, and anycombination thereof. In yet other embodiments, the marker nucleic acidis selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, SEQ ID NO:85 and any combination thereof.

In some embodiments, the marker nucleic acid is selected from the groupconsisting of PPR1_P5_1, PPR3_P4_2, PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9,CMSS03, CMSS10, CMSS15, CMSS34, DASCMS-SRf31, DASCMS-SRf34,DASCMS-SRf321, DASCMS-SRf39, and any combination thereof. In otherembodiments, the marker nucleic acid is selected from the groupconsisting of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and anycombination thereof. In yet other embodiments, the marker nucleic acidis selected from the group consisting of CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, DASCMS-SRf39, and anycombination thereof. In other embodiments, the marker nucleic acid isselected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and anycombination thereof.

In various embodiments, at least one of the markers is associated within8.3 Mb of the functional restorer gene for maize S-type cytoplasmic malesterility. In other various embodiments, at least one of the markers isassociated within 0.5 Mb of the functional restorer gene for maizeS-type cytoplasmic male sterility. In yet other various embodiments, atleast one of the markers is associated within 100 kb of the functionalrestorer gene for maize S-type cytoplasmic male sterility.

In some embodiments, the plant is a maize plant. In another embodiment,the maize plant belongs to the Stiff Stalk heterotic group.

In yet another aspect of the present disclosure, the method furthercomprises the steps of isolating nucleic acid molecules from thepopulation of progeny plants; contacting the isolated nucleic acidmolecules with a set of oligonucleotides; and amplifying the isolatednucleic acid molecules and the oligonucleotides to produce an amplicon,wherein the amplicon comprises a detectable signal that is indicative ofthe presence of the functional restorer gene for maize S-typecytoplasmic male sterility.

In one embodiment, a method for transferring an Rf3 gene into a progenyplant is provided. The method comprises the steps of (a) crossing afirst parent plant and a second parent plant to produce a progeny plant,wherein at least one parent plant comprises the Rf3 gene; (b) analyzingthe progeny plant for the presence of at least one marker that is linkedto the Rf3 gene to obtain an Rf3 progeny plant; (c) backcrossing the Rf3progeny plant with either the first parent plant or the second parentplant to produce a next-generation progeny plant; and (d) analyzing thenext-generation progeny plant for the presence of the at least onemarker that is linked to the Rf3 gene to obtain an Rf3 next-generationprogeny plant.

In some embodiments, the marker nucleic acid comprises a haplotype ofalleles linked to the functional restorer gene for maize S-typecytoplasmic male sterility. In other embodiments, the marker nucleicacid is selected from the group consisting of Mo17-14388, PZE-102180901,PZE-102180129, FG-1318, PZE-102182167, PZE-102182672, PZE-102182718,PZE-102183578, PZE-102183795, DAS-PZ-13844, PZE-102184593, PPR1_P5_1,PPR3_P4_2, PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9, CMSS03, CMSS10, CMSS15,CMSS34, DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, DASCMS-SRf39, and anycombination thereof. In yet other embodiments, the marker nucleic acidis selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, SEQ ID NO:85 and any combination thereof.

In some embodiments, the marker nucleic acid is selected from the groupconsisting of PPR1_P5_1, PPR3_P4_2, PPR8_P6_1, PPR3-5, PPR3-7, PPR3-9,CMSS03, CMSS10, CMSS15, CMSS34, DASCMS-SRf31, DASCMS-SRf34,DASCMS-SRf321, DASCMS-SRf39, and any combination thereof. In otherembodiments, the marker nucleic acid is selected from the groupconsisting of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and anycombination thereof. In yet other embodiments, the marker nucleic acidis selected from the group consisting of CMSS03, CMSS10, CMSS15, CMSS34,DASCMS-SRf31, DASCMS-SRf34, DASCMS-SRf321, DASCMS-SRf39, and anycombination thereof. In other embodiments, the marker nucleic acid isselected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and anycombination thereof.

In various embodiments, at least one of the markers is associated within8.3 Mb of the functional restorer gene for maize S-type cytoplasmic malesterility. In other various embodiments, at least one of the markers isassociated within 0.5 Mb of the functional restorer gene for maizeS-type cytoplasmic male sterility. In yet other various embodiments, atleast one of the markers is associated within 100 kb of the functionalrestorer gene for maize S-type cytoplasmic male sterility.

In some embodiments, the plant is a maize plant. In another embodiment,the maize plant belongs to the Stiff Stalk heterotic group. In yetanother embodiment, the Rf3 gene is SEQ ID NO:92.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. The examples should not be construed tolimit the disclosure to the particular features or embodimentsexemplified.

EXAMPLES Example 1: Plant Material

A male sterile line of CMS-S type, 4XP811-D (U.S. Pat. No. 7,135,629),and a male sterile restorer line responding to CMS-S type, LH60, wereused as parents to generate F₁ progeny. The F₁ progeny where then selfedto generate an F₂ population. The F₂ population, consisting of 450individuals, was used for identification of the Rf3 gene and markerslinked (e.g., linked; tightly linked; or extremely tightly linked) tothe Rf3 gene.

A BC₁ population of 275 individuals derived from 4XP811-D×MBB56, a malesterile restorer line responding to CMS-S type, was used for linkage mapanalysis with molecular markers designed from the 10 PPR genes withinthe 1.3 Mb interval.

Example 2: Fertility Classification

The individuals from the F₂ and BC₁ populations were phenotypicallyclassified according to pollen shed from the tassels. Plants that shedpollen were classified as fertile. Plants that did not shed pollen wereclassified as sterile. Partial fertile plants were observed in the F₂population but not in the BC₁ population.

The individuals from the BC₁ population were also phenotypicallyclassified by determining the vitality of pollen grain by using 1% KI-I₂staining. For the fertile plants, the staining results show well stainedpollen that were starch-filled and for the sterile plants, the stainingresults show un-stained collapsed pollen. Table 1 provides thesegregation data from the F₂ and BC₁ mapping populations.

TABLE 1 Phenotypic segregation data from the F₂ and BC₁ mappingpopulations Semi-fertile Non-pollinated Population Size Fertile plantsplants Sterile plants plants Broken plants F₂ 450 347 73 24 4 2 BC₁ 275139 0 120 0 16

Example 3: DNA Extraction and Quantification

Genomic DNA was extracted from 8 leaf punches per sample using theMagAttract™ DNA extraction method (Qiagen, Valencia, Calif.) and theBiocel 1800™ (Agilent Technologies, Santa Clara, Calif.). DNA sampleswere quantified with Quant-iT™ PicoGreen® Quantification Kit(Invitrogen, Carlsbad, Calif.) per manufacturer's instructions or withthe Nanodrop 8000 Spectrophotometer™ (Thermo Scientific, Rockford, Ill.)per manufacturer's instructions. The DNA concentration was normalized to6 ng/μL for use in the KASPar™ genotyping system (KBioscience Inc.,Hoddesdon, UK).

Example 4: KASPar™ SNP Genotyping System

The Competitive Allele-Specific PCR genotyping system (KASPar™) is a SNPdetection system that uses a technique based on allele-specific oligoextension and fluorescence resonance energy transfer (FRET) for signalgeneration. Each SNP marker in a KASPar™ assay requires only twocomponents: The assay mix (a mixture of three unlabelled primers: twoallele specific oligos, and one common reverse locus specific oligo);and the reaction mix (the other components required for PCR, includingthe universal fluorescent reporting system and Taq polymerase).Fluorescent signals after the completion of KASPar™ reactions are readin a spectrofluorometer with an excitation wavelength at 485 nm, and anemission wavelength at 535 nm for the FAM fluorophore; and an excitationwavelength at 525 nm, and an emission wavelength at 560 nm for the VICfluorophore. The data were analyzed using Klustercaller™ software(KBiosciences Inc.) to determine the genotypes of each SNP marker in apopulation.

The KASPar™ assays based on SNPs or InDels were designed with theKraken™ workflow manager (KBioSciences, Hoddesdon, Hertfordshire, UK).KASPar™ reactions were set up according to Tables 2 and 3. PCR cyclesstarted at 94° C. for 15 minutes, then 20 cycles with 10 seconds ofdenature at 94° C. and 5 seconds of annealing at 57° C., then 10 secondsof extension at 72° C., followed by 22 cycles with 10 seconds ofdenature at 94° C. and 20 seconds of annealing at 57° C., then 40seconds of extension at 72° C. ABI GeneAmp® PCR System 9700 (AppliedBiosystems, Foster City, Calif.) was used for amplification. PCRproducts were measured by PheraStar™ spectrofluorometer (BMG LABTECHInc., Cary, N.C.).

TABLE 2 Recipe for assay mix set up Concentration in Assay Mix Volume inAssay Mix (μM) (μl) Allele Specific Primer 1 (A1, 12 12 100 μM) AlleleSpecific Primer 2 (A2, 12 12 100 μM) Common (reverse) Primer (C1, 30 30100 μM) H₂0/TrisHCl (10 mM, pH 8.3) — 46 TOTAL 100

TABLE 3 Recipe for KASPar ™ reaction in a 5 μl final volume ComponentsVolume (μl) DNA (5 ng/μl): 1 2X Reaction Mix: 2.5 Assay Mix: 0.07 *MgCl2(50 mM): 0.04 H2O: 1.39 TOTAL: 5

Example 5: Preliminary Genetic Mapping of Rf3 in the F₂ Population UsingSNP Markers

JoinMap 4.0™ (Van Ooijen, J. W. et al, 2006) was used to create thegenetic linkage map and MapQTL 5.0™ (Van Ooijen, J. W. et al, 2006) wasused to map the QTLs in the F₂ population. The interval mapping methodwas performed. When a LOD score exceeds the significance threshold(permutation test results) on a linkage group, a segregating QTL isdetected; the position with the largest LOD on the linkage group is theestimated position of the QTL on the map.

KASPar™ assays were developed for 140 selected SNP markers (one markerper 10 cM for chromosome 2 and per 20 cM for the other chromosomes), andwere used to genotype the whole population for linkage map constructionand QTL analysis. One hundred thirty-three SNP markers were successfullyassigned to 10 linkage groups with a LOD threshold of 3.0.

A 3.6 LOD value was set as the significant threshold after running 1000permutations. Genome-wide QTL analysis detected one significant QTL forrestoring the CMS-S fertility of this germplasm (see FIG. 1). The QTLexplained 30% of the total phenotypic variation. This QTL was tightlylinked to molecular markers Mo17-14388, PZE-102180901, PZE-102180129,FG-1318, PZE-102182167, PZE-102182672, PZE-102182718, PZE-102183578,PZE-102183795, DAS-PZ-13844, and PZE-102184593 within a 8.3 Mb region onchromosome 2, and its location was co-incident with that of the restorergene Rf3 (see FIG. 1, Table 3).

TABLE 3 Exemplary markers that co-segregate with the Rf3 locus in the8.3 Mb region on chromosome 2. Allele Allele specific specific CommonSEQ Genetic primer 1 primer 2 primer ID Position % Additive SEQ ID SEQID SEQ ID Markers NO. (cM) LOD Variance Expl. Effect NO. NO. NO.Mo17-14388 1 129.25 29.5 57.46 26.6 −7.09 12 23 34 PZE- 2 131.14 31.8356.08 28.1 −7.36 13 24 35 102180901 PZE- 3 131.47 28.85 57.85 25.9 −6.9414 25 36 102180129 FG-1318 4 131.6 29.78 57.29 26.6 −7.09 15 26 37 PZE-5 133.47 30.54 56.84 27.2 −7.34 16 27 38 102182167 PZE- 6 133.59 30.4956.87 27.1 −7.33 17 28 39 102182672 PZE- 7 133.59 30.49 56.87 27.1 −7.3318 29 40 102182718 PZE- 8 135.77 31.87 56.06 28.1 −7.66 19 30 41102183578 PZE- 9 135.99 32.97 55.43 29 −7.83 20 31 42 102183795DAS-PZ-13844 10 136.36 32.24 55.85 28.4 −7.72 21 32 43 PZE- 11 136.5529.65 57.37 26.5 −7.33 22 33 44 102184593

Example 6: Annotation of the 8.3 Mb QTL Region

The annotation of the 8.3 Mb QTL region was performed from nucleotidesequence from 220,446,527 bp to 228,748,276 bp, using the Zea mays c.v.B73 Reference Genome version 2, which is publically available at suchsites as Maize GDB. A total of 142 genes were found in this region (datanot show). Ten of the 142 genes were PPR genes (identified withasterisks; ***) (Table 4). These 10 PPR genes were located within a1,282,382 by interval, where the Rf3 gene was mapped. Some of PPRsmatched known restorer genes from some previous studies, and could beRf3 candidate genes.

TABLE 4 Results of the annotation of the 8.3 Mb QTL region. Ten PPRgenes were identified in the region. The PPR genes are identified withasterisks; ***. Chr Start Stop Gene Chr2 226676462 226678841Pentatricopeptide repeat *** Chr2 226679089 226680887 Bifunctionalinhibitor/plant lipid transfer protein/seed storage Chr2 226699977226705433 BTB/POZ-like Chr2 226699977 226705433 Tetratricopeptide regionChr2 226699977 226705433 BTB/POZ fold Chr2 226699977 226705433Tetratricopeptide TPR-1 Chr2 226963057 227123377 Serine/threonineprotein kinase Chr2 226963057 227123377 Serine/threonine protein kinase,active site Chr2 226963057 227123377 Protein kinase-like Chr2 226963057227123377 Serine/threonine protein kinase Chr2 226963057 227123377Protein kinase-like Chr2 226963057 227123377 Protein kinase-like Chr2227029696 227031991 Homeobox Chr2 227029696 227031991 Homeodomain-likeChr2 227045894 227046871 Pentatricopeptide repeat *** Chr2 227209438227212064 Pentatricopeptide repeat *** Chr2 227213495 227214494 Proteinkinase-like Chr2 227264500 227268560 Helix-loop-helix DNA-binding Chr2227264500 227268560 Cyclin-like Chr2 227264500 227268560 Basichelix-loop-helix dimerisation region bHLH Chr2 227277494 227282413 KelchChr2 227277494 227282413 Galactose oxidase/kelch, beta- propeller Chr2227277494 227282413 Kelch repeat type 1 Chr2 227516080 227537665 Proteinkinase-like Chr2 227544895 227545712 Histone H2A Chr2 227544895227545712 Histone-fold Chr2 227544895 227545712 Histone core Chr2227586065 227587703 Pentatricopeptide repeat *** Chr2 227599623227600372 Pentatricopeptide repeat *** Chr2 227604054 227608803Pentatricopeptide repeat *** Chr2 227610560 227618077 DNA topoisomeraseI, C-terminal Chr2 227610560 227618077 DNA breaking-rejoining enzyme,catalytic core Chr2 227618518 227619348 Pentatricopeptide repeat ***Chr2 227652480 227654480 Protein kinase-like Chr2 227657388 227658767Histone H2A Chr2 227657388 227658767 Histone-fold Chr2 227657388227658767 Histone core Chr2 227876866 227880456 Pentatricopeptide repeat*** Chr2 227882663 227886563 Antifreeze protein, type I Chr2 227882663227886563 Antifreeze protein, type I Chr2 227893366 227895004Pentatricopeptide repeat *** Chr2 227932245 227946984 Histone H2A Chr2227932245 227946984 Histone-fold Chr2 227932245 227946984 Histone coreChr2 227955470 227957312 Protein kinase-like Chr2 227958844 227962673Pentatricopeptide repeat ***

Example 7: Fine Mapping the Rf3 Gene to a 1.3 Mb Region on Chromosome 2

After the PPR genes were identified based on the annotation of the QTLregion in B73 reference genome, homolog sequences from CMS-S line4XP811-D and restorer line LH60 were generated using PCR with specificprimers which were designed based on the PPR gene sequences in B73. ThePCR was performed in total volumes of 50 μl containing 100 ng of genomicDNA, 10× Qiagen™ PCR buffer, 25 mM MgCl₂, 2.5 mM of each dNTP, 0.25 mMof specific primers, and 5 U Taq plus polymerase. The PCR conditions forspecific primers were 2-minutes initial denaturation at 94° C., followedby 7 cycles of 98° C. for 10 seconds, 62° C. with −1.0° C. touchdown for20 seconds and 72° C. for 4 minutes 30 seconds, then followed by 25cycles of 98° C. for 10 seconds, 56° C. for 20 seconds and 72° C. for 4minutes 30 seconds, with a final extension at 72° C. for 10 minutes. PCRproducts were separated by 2% agarose e-gels and purified using a QiagenDNA purification Kit™ (Qiagen, Germantown, Md.). The purified ampliconswere cloned and sequenced by Eurofins MWG Operon (Huntsville, Ala.).

Since the PPR genes contain many repeat sequences, only a few PCRproducts were obtained from several primer sets that were designed toisolate PPR genes. Sequences from PCR products were aligned withSequencher 4.10.1™ software (Gene Codes, Ann Arbor, Mich.) between theCMS-S and restorer lines. Variations were identified in the alignedsequences and KASPar™ assays were designed to detect these variations.Six SNP markers polymorphic between parental lines 4XP811-D and LH60 areshown in Table 5.

TABLE 5 Polymorphic markers developed from several PPR genes AlleleAllele specific specific Common SEQ primer 1 primer 2 primer Marker SNPID NO. SEQ ID NO. SEQ ID NO. SEQ ID NO. PPR1_P5_1 G/C 45 51 57 63PPR3_P4_2 T/— 46 52 58 64 PPR8_P6_1 A/G 47 53 59 65 PPR3-5 A/G 48 54 6066 PPR3-7 T/— 49 55 61 67 PPR3-9 G/A 50 56 62 68

An F2 mapping population 4XP811-D×LH60 was used to map the sixpolymorphic markers with JoinMap 4.0 software. This population was usedto primarily identify the Rf3 restorer gene location on the long arm ofchromosome 2. The map contained 133 markers, which were evenlydistributed on the whole maize genome and the QTL was well defined andconfirmed. Six markers, PPR1_P5_1, PPR3-5, PPR3-7, PPR3-9, PPR8_P6-1,and PPR3_P4_2, were mapped in the peak region with highest LOD scores ofthe mapped QTL, which was 1.3 Mb in length.

Example 9: Whole Genome Sequencing and Gene-Specific Marker Development

To further identify the specific Rf3 restorer gene sequence and the genesequence of the rf3 mutation, which results in cytoplasmic malesterility, a whole genome sequencing approach was utilized. The twoCMS-S lines, 4XP811-D and 7SH382ms, and the two restorer lines, LH60 andMBB56, were used for sequencing analysis.

A total of 10 PPR genes (annotated sequentially as PPR 1-10) wereidentified on chromosome 2 within a 1.3 Mb genomic interval aftercomparison of the sequenced genomic regions with annotated sequence ofthe reference genome from Zea mays c.v. B73 (available at Maize GDB). Afull length coding sequence that corresponds with PPR2 did not exist inthe annotated B73 genome and could not be predicted as a full length,functional gene. As a result, the full gene sequence for PPR2 wasobtained from a sequence comparison to the reference genome of Zea maysc.v. Mo17 (available at Maize GDB). The resulting sequences for the 10PPR genes (PPR2 had two sequences, the first sequence from line B73 andthe second sequence from line Mo17) were assembled for referencesequence information based on whole genome sequencing data of the twoCMS-S lines and two restorer lines.

Next, the sequences were aligned between CMS-S lines, restorer lines,and the reference genomic sequences for all of the PPR genes. Anygenomic sequence variations were identified from the aligned genomicsequences of the PPR genes and noted.

Based on sequences of 10 PPR gene segments, 58 PCR primer pairs weredesigned and used to screen CMS-S and restorer lines. Only four primersets (Table 6) from PPR2 showed polymorphisms, which suggested that PPR2was associated with fertility restoration.

Based on the variations within the PPR2 gene, 21 KASPar™ assays weredeveloped and screened on the BC₁ mapping population, and three assaysshowed polymorphisms between the parental lines (Table 6).

TABLE 6 Polymorphic molecular markers from the PPR2 gene Primer 1 Primer2 Primer 3 Marker Marker Type SNP SEQ ID NO. SEQ ID NO. SEQ ID NO.CMSS03 PCR on e-gel T/C 69 70 n/a CMSS10 PCR on e-gel T/G 71 72 n/aCMSS15 PCR on e-gel T/A 73 74 n/a CMSS34 PCR on e-gel T/G 75 76 n/aDASCMS-SRf31 KASPar ™ T/G 77 80 83 DASCMS-SRf34 KASPar ™ C/G 78 81 84DASCMS-SRf321 KASPar ™ T/C 79 82 85

Example 10: PPR2 Gene Specific Markers Co-Segregate with Rf3 on LinkageMap

The BC₁ mapping population was genotyped using KASPar™ assays with 482SNP markers, which were evenly distributed on the whole genome. Fourhundred and thirty-seven markers (including 4 PCR-based markers fromPPR2, 432 SNP markers across the genome, and 1 marker based onsterile-fertile phenotype of the BC₁ mapping population) were used tocreate a genetic linkage with JoinMap 4.0™ software. With the geneticlinkage map, phenotypic data and genotypic data, a whole genome QTLanalysis was performed using MapQTL 6.0™ software (Kyazma B.V.,Netherlands). The results showed that all of the PPR2 gene specificmarkers (Tables 5 and 6) co-segregated with the Rf3 locus at a geneticposition of 177.08 cM, a LOD of 99.99, and explaining 100% of thephenotypic variation.

Example 11: Putative Rf3 Candidate Mutations and Expression Analysis ofthe Gene Coding PPR2 Protein by Real-Time PCR

To validate whether the expression of the Rf3-PPR2 protein is correlatedwith fertility restoration of CMS-S maize, a real-time PCR (RT-PCR) wasperformed to quantitatively determine the expression pattern of theRf3-PPR2 gene in fertile plants and the rf3-PPR2 gene in CMS plants.Several specific primer pairs and probes were designed based on portionsof the polynucleotide sequences that contained amino acids withvariations in the Rf3-PPR2 protein coding gene. Total RNA was extractedfrom two lines, 4XP811-D (cytoplasmic male sterile) and LH60 (restorerline), F3 individuals derived from an F2 ear segregating for the 1.3 Mbregion of long arm chromosome 2, and three commercial maize lines.

The plants to be analyzed via the Taqman® assay were grown in agreenhouse. Leaf tissues were collected from 7 week (just beforetasselling) and 10 week old plants (after pollination). Tassel tissueswith developing anthers/pollens and shed pollens (in fertile plants)were also collected. Total RNA was extracted using Qiagen RNeasy PlantMini Kit™ and cDNA was synthesized using Qiagen QuantiTect ReverseTranscription Kit™ (Qiagen, Carlsbad, Calif.). The expression levelswere quantitated by comparison to a maize internal control gene,elongation factor α-1 (EF α1). (Czechowski T, et al., Plant Physiol.,September; 139(1):5-17, 2005). Primers for Rf3-PPR2 and EF α1 and duallabeled probes with FAM or VIC dyes and Minor Groove Binding NonFluorescence Quencher™ I (MGBNFQ) quencher were synthesized by AppliedBiosystems (Foster City, Calif.). Taqman® genotyping master mix (AppliedBiosystems, Foster City, Calif.) was used to set up 10 μl PCR reactionsand the PCR was performed on Roche LightCycler 480™ thermocycler (Roche,Indianapolis, Ind.). The PCR program was initiated with 10 minutesactivation of the Taq enzyme at 95° C., followed by 50 cycles of 95° C.for 10 seconds and 58° C. for 38 seconds. Fluorescence signals wererecorded at the end of each cycle. Relative expression levels ofRf3-PPR2 to EF α1 was calculated using the Delta Delta CT method.

Seven Taqman® assays were designed based on mutations that resulted inamino acid changes within the Rf3-PPR2 gene. Only the DASCMS-SRf39 assayamplified an amplicon that corresponded with the presence of Rf3 or rf3gene sequence in the fertile restorer lines and cytoplasmic male sterilelines, respectively (Table 7). This assay was able to identify a singlemutation that resulted in the rf3 cytoplasmic male sterile phenotype. Assuch, this single mutation could be used to discern between the rf3cytoplasmic male sterile and the Rf3 restored fertile plants.Interestingly, Rf3 expression levels were distinguishable between Rf3homozygous (Rf3/Rf3) and heterozygous (Rf3/rf3) F3 individuals, whichcould explain why homozygous (Rf3/Rf3) plants shed 100% starched-filledfertile pollen while heterozygous (Rf3/rf3) plants shed approximately50% starch-filled fertile pollen. Finally, the Rf3-PPR2 gene, whichrestores S-type CMS cytoplasm, is expressed in both tassel tissuecontaining immature pollen, and leaf tissue.

TABLE 7 Primers and probes used for the DASCMS-SRf39 RT-PCR assay andEF α1 internal control. Reaction Primer Name Primer Sequence SEQ ID NO:Rf3 allele Forward Primer GTACTCATGGTAGTTTACTGAAAGCCA 86 specific:Reverse Primer GCATCCATTACCCTTCCCAAT 87 DASCMS- ProbeATCTTGACATTGTTTTATTCAGTTCG 88 SRf39 Internal ForwardATAACGTGCCTTGGAGTATTTGG 89 Control: Primer Elongation ReverseTGGAGTGAAGCAGATGATTTGC 90 Factor α-1 Primer Probe TTGCATCCATCTTGTTGC 91

What is claimed is:
 1. A method for restoring fertility in a maizeplant, the method comprising the steps of: (a) crossing a female maizeplant with a male maize plant to generate a population of progeny maizeplants, wherein the female maize plant is an S-type cytoplasmic malesterile maize plant, and wherein the male maize plant possesses afunctional Rf3 restorer gene for S-type cytoplasmic male sterility; (b)screening the population of progeny maize plants to identify a fertileprogeny maize plant comprising marker nucleic acid Mo17-14388 comprisingan allele linked to the functional Rf3 restorer gene for maize S-typecytoplasmic male sterility by extracting nucleic acids from the progenyplants and detecting the marker nucleic acid Mo17-14388; (c) selectingthe fertile progeny maize plant comprising the marker nucleic acidMo17-14388 comprising the allele linked to the functional Rf3 restorergene for maize S-type cytoplasmic male sterility; and (d) propagatingthe fertile progeny maize plant, wherein the fertile progeny maize plantcomprises the functional Rf3 restorer gene for maize S-type cytoplasmicmale sterility.
 2. A method for restoring fertility in a maize plant,the method comprising the steps of: (a) crossing a female maize plantwith a male maize plant to generate a population of progeny maizeplants, wherein the female maize plant is an S-type cytoplasmic malesterile maize plant, and wherein the male maize plant possesses afunctional Rf3 restorer gene for S-type cytoplasmic male sterility; (b)screening the population of progeny maize plants to identify a fertileprogeny maize plant comprising marker nucleic acid DASCMS-SRf39comprising an allele linked to the functional Rf3 restorer gene formaize S-type cytoplasmic male sterility by extracting nucleic acids fromthe progeny plants and detecting the marker nucleic acid DASCMS-SRf39;(c) selecting the fertile progeny maize plant comprising the markernucleic acid DASCMS-SRf39 comprising the allele linked to the functionalRf3 restorer gene for maize S-type cytoplasmic male sterility; and (d)propagating the fertile progeny maize plant, wherein the fertile progenymaize plant comprises the functional Rf3 restorer gene for maize S-typecytoplasmic male sterility.
 3. The method of claim 1, wherein the markernucleic acid Mo17-14388 is selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 12, and SEQ ID NO: 23, and any combination thereof. 4.The method of claim 2, wherein the marker nucleic acid DASCMS-SRf39 isselected from the group consisting of SEQ ID NO: 86, SEQ ID NO: 87, andSEQ ID NO: 88, and any combination thereof.
 5. The method of claim 1,wherein the marker nucleic acid Mo17-14388 is located on chromosome 2within 8.3 Mb of the functional Rf3 restorer gene for maize S-typecytoplasmic male sterility.
 6. The method of claim 1, the method furthercomprising introducing the functional Rf3 restorer gene for maize S-typecytoplasmic male sterility into the male plant.
 7. The method of claim6, wherein the introducing is selected from the group consisting oftransformation, crossing, backcrossing, homologous recombination, andmutagenesis.
 8. The method of claim 1, wherein the progeny maize plantis a hybrid maize plant.
 9. The method of claim 8, wherein the hybridmaize plant belongs to the Stiff Stalk heterotic group.
 10. The methodof claim 1, said screening comprising the steps of: isolating nucleicacid molecules from the population of progeny maize plants; contactingthe isolated nucleic acid molecules with a set of oligonucleotides; andamplifying the isolated nucleic acid molecules and the oligonucleotidesto produce an amplicon, wherein the amplicon comprises a detectablesignal that is indicative of the presence of the functional Rf3 restorergene for maize S-type cytoplasmic male sterility.
 11. The method ofclaim 6, wherein the functional Rf3 restorer gene for maize S-typecytoplasmic male sterility consists essentially of SEQ ID NO:
 92. 12.The method of claim 11, wherein the functional Rf3 restorer gene formaize S-type cytoplasmic male sterility is comprised in an organismselected from the group consisting of a plant, a yeast, a bacterium, anda virus.
 13. The method of claim 12, wherein the functional Rf3 restorergene for maize S-type cytoplasmic male sterility is comprised in a plantcell.
 14. The method of claim 9, wherein the female maize plant or themale maize plant of the hybrid maize plant belongs to the Stiff Stalkheterotic group.
 15. The method of claim 2, wherein the marker nucleicacid DASCMS-SRf39 is located on chromosome 2 within 8.3 Mb of thefunctional Rf3 restorer gene for maize S-type cytoplasmic malesterility.
 16. The method of claim 2, wherein the marker nucleic acidDASCMS-SRf39 is located on chromosome 2 within 1.3 Mb of the functionalRf3 restorer gene for maize S-type cytoplasmic male sterility.
 17. Themethod of claim 2, wherein the marker nucleic acid DASCMS-SRf39 islocated on chromosome 2 within 0.5 Mb of the functional Rf3 restorergene for maize S-type cytoplasmic male sterility.
 18. The method ofclaim 2, wherein the marker nucleic acid DASCMS-SRf39 is located onchromosome 2 within 100 kb of the functional Rf3 restorer gene for maizeS-type cytoplasmic male sterility.
 19. The method of claim 2, the methodfurther comprising introducing the functional Rf3 restorer gene formaize S-type cytoplasmic male sterility into the male plant.
 20. Themethod of claim 19, wherein the introducing is selected from the groupconsisting of transformation, crossing, backcrossing, homologousrecombination, and mutagenesis.
 21. The method of claim 2, wherein theprogeny maize plant is a hybrid maize plant.
 22. The method of claim 21,wherein the hybrid maize plant belongs to the Stiff Stalk heteroticgroup.
 23. The method of claim 2, said screening comprising the stepsof: isolating nucleic acid molecules from the population of progenymaize plants; contacting the isolated nucleic acid molecules with a setof oligonucleotides; and amplifying the isolated nucleic acid moleculesand the oligonucleotides to produce an amplicon, wherein the ampliconcomprises a detectable signal that is indicative of the presence of thefunctional Rf3 restorer gene for maize S-type cytoplasmic malesterility.
 24. The method of claim 19, wherein the functional Rf3restorer gene for maize S-type cytoplasmic male sterility consistsessentially of SEQ ID NO:
 92. 25. The method of claim 24, wherein thefunctional Rf3 restorer gene for maize S-type cytoplasmic male sterilityis comprised in an organism selected from the group consisting of aplant, a yeast, a bacterium, and a virus.
 26. The method of claim 25,wherein the functional Rf3 restorer gene for maize S-type cytoplasmicmale sterility is comprised in a plant cell.
 27. The method of claim 22,wherein the female maize plant or the male maize plant of the hybridmaize plant belongs to the Stiff Stalk heterotic group.